Transmissible spongiform encephalopathy (TSE) agents induce fatal neurodegenerative diseases in mammalian species and humans. The TSE group in animals includes scrapie in sheep and goat, bovine spongiform encephalopathy (BSE) in cattle, feline spongiform encephalopathy, transmissible mink encephalopathy, and chronic wasting disease (CWD) in wild ruminants. Among them, BSE, commonly known as mad cow disease, has brought enormous economic consequences since its first incidence in the United Kingdom in 1986. The BSE outbreaks peaked in the United Kingdom in 1993 at almost 1000 new cases per week, and it caused more than 182,000 cases between 1988 and 2002. In addition, the emergence of a new variant form of Creutzfeldt-Jakob Disease (vCJD) in humans in the United Kingdom has been proposed to be possibly linked with BSE.
Meat and bone meal, an ingredient of animal feed, contaminated with a TSE agent was believed to be the major vehicle of BSE transmission, according to epidemiological inquiry. Meat and bone meal has been produced by rendering the discarded animal fat, bones, offal, and whole carcasses from bovine, ovine, porcine, and poultry. Although the use of meat and bone meal in cattle as a nitrogen supplement had been a common practice for several decades, changes in rendering operations in the 1970s and 1980s may have allowed the survival of the contagious agents that can be transmitted to the cattle through the meat and bone meal. The oral route was the major mode of natural transmission of BSE to cattle. To prevent the spread of BSE, the European Union in 1988 banned the inclusion of ruminant-derived proteins in animal feed. The U.S. Food and Drug Administration also introduced the feed ban in 1997 to prohibit the use of proteins derived from mammalian tissues in feeding ruminants. Nevertheless, the use of mammalian blood and blood products and any products having only protein of porcine or equine origin in ruminant feed products is still allowed. However, recent evidence indicates that blood also carries some level of infectivity for TSE since transmission of TSE has been demonstrated through inoculation of blood in animals infected with various strains of TSE (see, e.g., Castilla et al., Nat. Med., Vol. 11, pp. 982-985, 2005; Houston et al., Lancet, Vol. 356, pp. 999-1000, 2000; and Taylor et al., J. Hosp. Infect., Vol. 46, pp. 78-79, 2000). In addition to being used in animal feed, animal blood is used as a source of human food in many countries, usually in the form of blood sausages, pudding, soup, bread and crackers. Even in the U.S., blood is used, e.g., in sausage products to enhance color, in bakery products as an egg substitute or in liquid foods as a clarifying agent.
Although prohibited, meat and bone meal from ruminant origin may still enter cattle diets accidentally as a result of cross-contamination during feed mixing at the feed mills, transportation, storage, or the farm. Indeed, the U.S. Food and Drug Administration found very low levels of prohibited meat and bone meal residues in the feedlot resulting from misformulation of the animal feed supplement at feed mills. In addition, although bovine blood and plasma are currently acceptable for certain uses, their future is uncertain due to changing attitudes of producers, blenders, and consumers who would like to have products that are “free” of bovine blood and plasma products. Feed blenders and manufacturers usually acquire these blood and plasma products from renderers who process or transport products from both ruminants (cattle, buffalo, sheep, goats, deer, elk, and antelopes) and nonruminants (pigs, and horses). Thus, care must be taken to ensure that cross-contamination does not occur in the blending or manufacturing process.
Tools that permit enforcement of the meat and bone meal bans to eradicate BSE are becoming increasingly important for compliance with animal byproduct regulation. Furthermore, the accurate labeling of meat products is mandated and monitored by the United States Department of Agriculture (USDA) as well as by state and local governments. Mixing undeclared species in meat products is illegal under Food Labeling Regulations.
To date, there are technical limitations in detecting prohibited residues in animal feed because most of the analytical methods cannot distinguish between allowable and prohibited bovine materials. Several methods have been developed to identify meat species including electrophoresis, chromatography, DNA hybridization, and immunoassays. Immunological techniques, including agar-gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) are most commonly applied for meat species identification.
There are several disadvantages to the AGID method. Concentrated antiserum preparations are required to obtain visible precipitin lines in AGID. Obtaining the antiserum is expensive in large-scale testing. Furthermore, the sensitivity of AGID is variable. Usually ten percent or more contamination must be present to detect adulteration with this method. Lastly, AGID cannot be used for species identification in cooked meat because of the shortage of commercial antiserum specific to cooked meats.
With respect to blood protein detection, the currently used methods such as spectrophotometry, the Takayama confirmatory test and immunochemical methods also exhibit several limitations. This is in part due to the fact that these assays are mainly based on heat-labile blood proteins, resulting in lack of species specificity and making them unusable for detection of blood in heat-treated samples such as cooked food or blood meal in animal feed.